The molecular target of many naturally-occurring antibiotics, including fosfomycin, cycloserine and β-lactams, is the synthesis of the bacterial cell wall. The frequency with which these types of antibiotics arose in evolution indicates that the pathway of cell wall biosynthesis is a particularly effective point of attack against bacteria. Genetic studies confirm the soundness of this process as a target, as temperature-sensitive alleles of the intracellular pathway genes are lytic, and therefore lethal. Since the building blocks of the cell wall are highly conserved structures in both Gram-positive and Gram-negative bacteria, but are unique to the eubacteria, novel inhibitors of cell wall formation are expected to be both broad spectrum and safe antibiotics.
The bacterial cell wall is a polymer, a single molecule composed of peptidoglycan that defines the boundary and shape of the cell. Assembled by crosslinking glycan chains with short peptide bridges (Rogers, H. J., H. R. Perkins, and J. B. Ward, 1980, Biosynthesis of peptidoglycan. p. 239-297. In Microbial cell walls and membranes. Chapman & Hall Ltd. London), the completed structure is strong enough to maintain cell integrity against an osmotic pressure differential of over four atmospheres, but also flexible enough to allow the cell to move, grow and divide.
The construction of the peptidoglycan begins in the cytoplasm with an activated sugar molecule, UDP-N-acetylglucosamine. After two reactions (catalyzed by MurA and MurB) that result in the placement of a lactyl group on the 3-OH of the glucosamine moiety, a series of ATP-dependent amino acid ligases (MurC, -D, -E, and -F) catalyze the stepwise synthesis of the pentapeptide sidechain using the newly synthesized lactyl carboxylate as the first acceptor site. After attachment of the sugar pentapeptide to a lipid carrier in the plasma membrane, another glucosamine unit is added to the 4-OH of the muramic acid moiety. The completed monomeric building block is moved across the membrane into the periplasm where the penicillin-binding proteins enzymatically add it into the growing cell wall (Lugtenberg, E. J. J., 1972, Studies on Escherichia coli enzymes involved in the synthesis of Uridine Diphosphate-N-Acetyl-Muramyl-pentapeptide. J. Bacteriol. 110:26-34; Mengin-Lecreulx, D., B. Flouret, and J. van Heijenoort, 1982, Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli. J. Bacteriol. 151: 1109-1117).
Because the pentapeptide sidechain is not synthesized ribosomally it contains more diverse chemical functionality than a typical peptide, both structurally and stereochemically. Two of the enzymes catalyze the addition of D-amino acids (MurD and MurF) and MurE mediates the formation of a peptide bond between the g-carboxylate of D-glutamate and the amino group of L-lysine. Presumably these structures render the exposed peptidoglycan resistant to the action of proteases, but they also imply that the active sites of the enzymes must have unusual structures in order to handle the somewhat uncommon substrates. These unusual active sites are targets to bind novel inhibitors that can have antimicrobial activity.
Among these potential enzyme targets is MurD. The first partial purification and characterization of a D-glutamate-adding enzyme was from Staphlococcus aureus (Ito, E. and J. L. Strominger, 1962. Enzymatic synthesis of the peptide in bacterial uridine nucleotides: Enzymatic addition of L-alanine, D-glutamic acid, and L-lysine. J. Biol. Chem. 237: 2689-2695; Nathenson, S. G., J. L. Strominger, and E. Ito, 1964. Enzymatic synthesis of the peptide in bacterial uridine nucleotides: purification and properties of D-Glutamic acid-adding enzyme, J. Biol. Chem. 239: 1773-1776), followed by studies in more detail on the isolated Eschericia coli enzyme (Blanot, D., A. Kretsovali, M. Abo-Ghalia, D. Mengin-Lecreulx, and J. van Heijenoort, 1983. Synthesis of analogues of precusors of bacterial peptidoglycan. In Peptides. Blaha, K. and P. Malon, eds. pp. 311-314, Walter de Gryter and Co. Berlin, N.Y.; Jin, H., Emanuele, J. J., Jr., Fairman, R., Robertson, J. G., Hail, M. E., Ho, H.-T., Falk, P. and Villafranca, J. J., 1996. Structural studies of Escherichia coli UDP-N-acetylmuramate: L-alanine ligase. Biochemistry 35: 14423-14431; Ito E. and J. L. Strominger, 1973. Enzymatic synthesis of the peptide in bacterial uridine nucleotides: Comparative biochemistry. J. Biol. Chem. 248: 3131-3136; Michaud, C. D. Blanot, B. Flouret, and J. van Heijenoort, 1987. Partial purification and specificity studies of the D-glutamate-adding and D-alanyl-D-alanine-adding enzymes from Escherichia coli K12. Eur. J. Biochem. 166: 631-637). A purified recombinant E. coli MurD was reported (Pratviel-Sosa F, D. Mengin-Lecreulx and J. van Heijenoort, 1991. Over-production, purification and properties of the uridine diphosphate N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli. Eur. J. Biochem. 202 (3):1169-1176) and genes encoding MurD have been cloned from several species of bacteria including E. coli (Ikeda, M., M. Wachi, F. Ishino, and M. Matsuhashi, 1990a. Nucleotide sequence involving murD and an open reading frame ORF-Y spacing murF and ftsW in Escherichia coli. Nucleic Acids Res. 18:1058; Mengin-Lecreulx, D., C Parquet, L. Desviat, J. Pla, B. Flouret, J. Ayala and J. van Heijenoort, 1989. Organization of the murE-murG region of Escherichia coli: Identification of the murD gene encoding the D-glutamic-acid-adding enzyme. J. Bacteriol. 171: 6126-6134) and Bacilus subtilis (Daniel, R. A., and J. Errington, 1993. DNA sequence of the murE-murD region of Bacillus subtilis 168. J. Gen. Microbiol. 139:361-370; Henriques, A. O. de Lencaster, H. and P. J. Piggot, 1992, A Bacillus subtilis morphogene cluster that includes spoVE is homologous to the mra region of Escherichia coli. Biochimie. 74: 735-748). More recently, Purified recombinant MurD enzymes were purified from Gram-positive cocci (El-Sherbeini, M., Geissler, W., Pittman, J., Yuan, X., Wong, K. K. and Pompliano, D. L. 1998, Cloning and expression of Staphylococcus aureus and Streptococcus pyogenes murD genes encoding uri dine diphosphate N-acetylmuramoyl-L-alanine:D-glutamate ligases. Gene, 210: 117-125).
Compounds have been designed and synthesized that have inhibitory activity against the E. coli enzyme (Tanner, M. E., S. Vaganay, van Heijenoort, J., and D. Blanot, 1996. Phosphinate Inhibitors of the D-Glutamic Acid-Adding Enzyme of Peptidoglycan Biosynthesis. J. Org. Chem. 61: 1756-1760), although they do not have antibacterial activity.